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作物学报 ›› 2018, Vol. 44 ›› Issue (12): 1793-1801.doi: 10.3724/SP.J.1006.2018.01793

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜芸薹素唑耐受因子(BnaBZR1/BnaBES1)全长CDS克隆与生物信息学分析

冯韬,官春云()   

  1. 湖南农业大学农学院 / 国家油料改良中心湖南分中心, 湖南长沙 410128
  • 收稿日期:2018-05-03 接受日期:2018-08-20 出版日期:2018-12-12 网络出版日期:2018-09-19
  • 通讯作者: 官春云
  • 基金资助:
    本研究由国家重点基础研究发展计划(973计划)项目资助(2015CB150206)

Cloning and Characterization of Brassinazole-resistant (BnaBZR1 and BnaBES1) CDS from Brassica napus L.

Tao FENG,Chun-Yun GUAN()   

  1. College of Agronomy, Hunan Agricultural University / National Oilseed Crops Improvement Center in Hunan, Changsha 410128, Hunan, China
  • Received:2018-05-03 Accepted:2018-08-20 Published:2018-12-12 Published online:2018-09-19
  • Contact: Chun-Yun GUAN
  • Supported by:
    This study was supported by the National Basic Research Program (973 Program).(2015CB150206)

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摘要：

芸薹素唑耐受因子(brassinazole-resistant, BZR)是油菜素内酯信号转导过程中的关键转录因子, 目前已知BZR1与BZR2 (BES1)两种亚型。本文在甘蓝型油菜湘油15号cDNA中克隆到3个BZR全长编码序列(coding sequence, CDS), 经比对鉴定分别为定位于A07染色体的1拷贝BZR1和定位于A06染色体的2拷贝BES1, 分别命名为BnaBZR1_A07、BnaBES1_A06F和BnaBES1_A06R, 序列长分别为996、993和996 bp, 各自编码331、330、331个氨基酸。这3个基因编码蛋白具有典型植物BZR/BES结构域, 亚细胞定位预测主要位于细胞核。多序列比对和进化分析表明, BnaBZR1/BnaBES1基因编码蛋白与甘蓝、白菜、拟南芥、亚麻芥等BZR/BES蛋白具有较高的同源性, 且同源物种间BZR1或BES1相似度高于同一物种或者近缘物种中BZR1与BES1间相似度, 表明BZR1与BES1分化是一个早期进化事件。湘油15号中这3个基因表达规律相似, 苗期和花期具有高水平的BnaBZR基因表达, 抽薹期和角果成熟期表达稍低; 且BnaBZR基因表达水平在地上部分的叶、茎、花和角果中相对于地下部分的根中较高。

关键词: 甘蓝型油菜, 芸薹素唑耐受因子, 基因克隆, 基因表达, 生物信息学分析

Abstract:

Brassinazole-resistant (BZR) is a key transcription factor in brassinosteroid signaling pathway of plants, containing brassinazole-resistant 1 (BZR1) and brassinazole-resistant 2 (BES1). In this study, three novel BZR1 coding sequences (CDSs) were isolated from cDNA of Brassica napus L. cv. Xiangyou 15 leaves, which were mapped on the chromosomes A07, A06, and A06, and designated as BnaBZR1_A07, BnaBES1_A06F, and BnaBES1_A06R, respectively. These three BnaBZR CDSs were 996, 993, and 996 bp in length and encoded predicted proteins with 331, 330, and 331 amino acid residues, respectively. BnaBZR proteins were predicted to be located on the cell nucleus and have a typical plant BZR/BES conserved domain. Multiple sequence alignments and phylogenetic analysis showed that the deduced amino acid sequences of BnaBZR were highly homologous to previously reported BZR/BES of Brassica oleracea, Arabidopsis thalian, and Eruca sativa. And the similarity of BZR1 or BES1 among different related species was higher than the similarity between BZR1 and BES1 in the same species or related species, indicating that BZR1 and BES1 differentiation is an early evolutionary event. The expression patterns of BnaBZR1_A07, BnaBES1_A06F, and BnaBES1_A06R in Xiangyou 15 were similar, whit high expression level at the seedling stage and flowering stage, while slightly lower level at the stage of bolting and podgrain ripening. The expression level of BnaBZR in the aerial parts such as leaves, stems, flowers and pods was higher than that in underground parts.

Key words: Brassica napus L., BZR, gene clone, gene expression, bionformatic analysis

图1

BnaBZR1_A07、BnaBES1_A06F和BnaBES1_A06R 基因克隆 M: 2K DNA marker; 1: BnaBZR1_A07编码序列; 2: BnaBES1_A06F编码序列; 3: BnaBES1_A06R编码序列。"

表1

BnaBZR1、BnaBES1_F和BnaBES1_R蛋白信息汇总表"

蛋白编号
Serial number		 氨基酸残基数
Base of amino acid residues		 摩尔质量
Molar mass (Da)		 等电点
Isoelectric point
BnaBZR1_A07 331 35 895.131 9.145
BnaBES1_A06F 330 36 071.176 9.745
BnaBES1_A06R 331 36 177.322 9.745

图2

BnaBZR1_A07、BnaBES1_A06F和BnaBES1_A06R蛋白二级结构"

表2

BnaBZR1_A07、BnaBES1_A06F和BnaBES1_A06R蛋白特殊识别位点序列总表"

蛋白编号
Serial number		 位点类型
Domains type		 数量
Number		 位置
Location		 序列
Sequence		 序列模式
Sequence motif
BnaBZR1_A07 PS00001 2 142-145, 143-146 NNSS, NSST N-{P}-[ST]-{P}
PS00004 1 21-24 RKPS [RK](2)-x-[ST]
PS00005 5 24-26, 84-86, 162-164, 179-181, 221-223 SWR, TYR, SLR, TSK, THR [ST]-x-[RK]
PS00006 4 24-27, 236-239, 230-233, 240-243 SWRE, SRGE, TIPE, STVD [ST]-x(2)-[DE]
PS00008 4 5-10, 154-159, 155-160, 325-330 GATSTS, GGIPSS, GIPSSSL, GNGKAR G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}
BnaBES1_A06F PS00001 1 138-141 NIST N-{P}-[ST]-{P}
PS00004 1 20-23 RKPS [RK](2)-x-[ST]
蛋白编号
Serial number		 位点类型
Domains type		 数量
Number		 位置
Location		 序列
Sequence		 序列模式
Sequence motif
BnaBES1_A06R PS00005 5 18-20, 23-25, 83-85, 99-101, 174-176 TRR, SWR, TYR, SSR, TNK [ST]-x-[RK]
PS00006 6 23-26, 112-115, 134 -137, 227 -230, 237-240, 305-308 SWRE, SPFE, SRGD, TIPE, STVD, TPWE [ST]-x(2)-[DE]
PS00008 5 5-10, 149-154, 150-155, 259-264, 325-330 GATSTS, GGIPSS, GIPSSL, GVSSAV, GNAKGR G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}
PS00016 1 135-137 RGD R-G-D
PS00001 1 139-142 NIST N-{P}-[ST]-{P}
PS00004 1 21-24 RKPS [RK](2)-x-[ST]
PS00005 5 18-20, 24-26, 84-86, 100-102, 175-177 TRR, SWR, TYR, SSR, TSK [ST]-x-[RK]
PS00006 6 24-27, 113-116, 135-138, 228-231, 238-241, 306-309 SWRE, SPFE, SRGD, TIPE, STVD, TPWE [ST]-x(2)-[DE]
PS00008 5 5-10, 150-155, 151-156, 260-265, 326-331 GATSTS, GGIPSS, GIPSSL, GVSSAV, GNAKGR G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}
PS00016 1 136-138 RGD R-G-D

图3

BnaBZR氨基酸序列比对"

图4

不同植物BZR/BES蛋白的进化树分析"

图5

不同发育时期不同组织中BnaBZR1_A07、BnaBES1_A06F和BnaBES1_A06R的表达量 DAG表示种子萌发后天数。"

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